Project summary The transforming human gammaherpesviruses EBV and KSHV establish stable latent infections in B cells, providing a lifelong reservoir of virus that can contribute to the development of malignant disease. Thus, defining the mechanisms that govern long-term latency is critical for designing rational strategies to prevent disease. In vivo studies of gammaherpesviruses in humans have been severely limited by the difficulties of working in the natural host. Murine gammaherpesvirus 68 (MHV68) is related to EBV and KSHV and causes lymphomas and lymphoproliferative disease in mice, providing a readily manipulable small animal model for mechanistic studies of the virus/host relationship in vivo. Like EBV and KSHV, MHV68 expresses lncRNAs whose functions during infection and pathogenesis are largely unknown. As virus-encoded lncRNAs are abundantly expressed in vivo in B cells during long-term latency and in hyperplastic B cell lesions during lymphoproliferative disease, we hypothesize that these lncRNAs play key roles in latency and lymphomagenesis. In support of this, our new data demonstrates that the MHV68 TMER4 transcript acts a unique lncRNA that is essential for latency and pathogenesis. Using a combination of cutting-edge high throughput sequencing approaches and a novel bioinformatic pipeline, we have now generated a comprehensive map of validated MHV68 lncRNA transcripts. With results from this discovery phase in hand, we will now test the hypothesis that MHV68 lncRNAs are essential for latency and tumorigenesis. We will: 1) Determine the roles of high priority MHV68 lncRNAs during in vivo infection; 2) Define the molecular mechanism by which lncRNA TMER4 facilitates latent infection, and determine the tripartite regulatory relationship between the novel M3M2 lncRNA, antisense TMERs/miRNAs and latency protein M2, and 3) Determine the role of virus and host lncRNAs in lymphomagenesis. The use of new genomic, bioinformatic and mutagenesis technology, in conjunction with systematic in vivo analyses of MHV68 lncRNA mutants, provides an extremely powerful means to determine the molecular mechanism by which lncRNAs contribute to gammaherpesvirus infection and lymphomagenesis in vivo. Further, the unique collaborative nature of this program project should allow us to define the contribution of common lncRNA-targeted pathways to latency and tumorigenesis.